PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA.

Abstract

Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic dulfide isomerase DsbA. This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual Intrasheet disulfide bond between the last two (.strands of its CD4-like carboxyl-terminal domain. The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo. Interestingly, the absence of the disuifide bond did not prevent PapD from folding in vito or from forming a complex with the pilus adhesin in vitro. We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperonelike fashion. Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD. However, the ability ofpilus subunits to achieve native-like conformations in vivo depended on PapD. These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

“…Our results, together with previous reports 13,23,24,48 , suggest a straightforward model for the FimC-catalyzed subunit folding of FimA and other pilus subunits. As the FimA fold has a high topological complexity (contact order) 28 , this might be the dominant factor determining the high kinetic barrier of spontaneous FimA folding.…”

“…In the case of the homologous P pilus system, pilus assembly was also abolished in a strain lacking DsbA. The effect of the dsbA deletion on P pilus assembly had been interpreted such that the single structural disulfide bond between Cys228 and Cys233 in the P pilus chaperone PapD would no longer form in the absence of DsbA, thereby preventing PapD folding and thus pilus biogenesis 23 . The results obtained in this study reveal that disulfide bond formation has a much more profound function in pilus assembly than previously assumed.…”

“…The deletion of the dsbA gene on the E. coli genome leads to the complete absence of type 1 pili 22 . Only when the entire type 1 pilus gene cluster was overexpressed via a plasmid in a dsbA deletion strain could type 1 pilus biogenesis still be observed, but at reduced levels compared to wild-type cells 23,24 . The latter finding does not contradict the dependence on DsbA of type 1 pilus biogenesis in wild-type E. coli strains and may be explained by partial oxidation of overexpressed pilus subunits by air oxygen, which is possibly catalyzed by traces of transition metals such as copper 45 .…”

“…The periplasmic disulfide oxidoreductase DsbA is the principal oxidant of periplasmic proteins with structural disulfide bonds 20,21 . DsbA is essential for the in vivo assembly of pili and other disulfide-bonded virulence factors, as deletion of the dsbA gene from E. coli abolishes pilus biogenesis [22][23][24] .…”

Chaperone Activity of DsbC

“…In fact, DsbA has been suggested to function as a chaperone in the maturation of secreted virulence factors of Vibrio cholerae (20) and in pilus biogenesis of Escherichia coli. (21). The high-resolution crystal structure analysis of DsbA (22) has shown that an extensive uncharged surface surrounding the active-site disulfide is responsible for peptide binding.…”